Effects of a Multi-Ingredient Longevity Formulation (DoNotAge®) on Whole Blood NAD+ Levels: A 28-Day Randomised, Double-Blind, Placebo-Controlled Trial

1. Introduction

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme found in every living cell, serving as a central mediator of cellular energy metabolism, DNA repair, chromatin remodelling, and sirtuin-dependent signalling pathways [1]. NAD+ levels decline progressively with age, and this decline has been implicated in mitochondrial dysfunction, impaired DNA repair, chronic inflammation, and metabolic deterioration [2]. Restoring NAD+ levels is therefore a primary target of longevity interventions, with the hypothesis that maintaining youthful NAD+ concentrations may slow or partially reverse multiple hallmarks of ageing.

Nicotinamide mononucleotide (NMN), a direct biosynthetic precursor to NAD+, has demonstrated the ability to elevate NAD+ levels in several human trials [3,4]. However, NMN is typically studied in isolation and at a single dose, while the biology of ageing involves multiple interconnected pathways. There is growing interest in whether multi-ingredient formulations targeting several hallmarks simultaneously may produce additive or synergistic effects on NAD+ metabolism [5].

DoNotAge® is a daily sachet formulation containing 15 bioactive ingredients, including NMN, trans-resveratrol, and three proprietary compounds (SulforaBoost®, SIRT6Activator®, Nitralis®). Several of these ingredients have established mechanistic links to NAD+ metabolism. Trans-resveratrol activates sirtuins, which are NAD+-dependent deacetylases [6]. Trimethylglycine (TMG) supports the methylation cycle, which is coupled to NAD+ biosynthesis [7]. Calcium alpha-ketoglutarate (Ca-AKG) participates in the TCA cycle and may influence NAD+/NADH ratios [8]. Quercetin has been shown to modulate NAD+ salvage pathway enzymes in preclinical models [9].

A prior 60-day randomised controlled trial conducted in Dubai demonstrated that DoNotAge® produced significant improvements in subjective well-being (WHO-5: +26.2 vs placebo, p < 0.0001) and fatigue (FACIT-Fatigue: +4.9 vs placebo, p < 0.0001) [10]. However, that trial relied on patient-reported outcomes. The present study was designed to complement those findings by measuring an objective molecular biomarker, whole blood NAD+, to determine whether the subjective benefits observed in the Dubai trial are accompanied by measurable changes in NAD+ biology.

The primary objective was to assess the change in whole blood NAD+ concentration from baseline to Day 28 in healthy adults receiving DoNotAge® compared with matched placebo.

2. Materials and Methods

2.1. Study Design

This was a 28-day, single-centre, randomised, double-blind, placebo-controlled, parallel-group trial conducted at LifeLab1, Vilnius, Lithuania. Participants were randomly allocated in a 1:1 ratio to receive either DoNotAge® (Group B) or a matched placebo (Group A). NAD+ measurements were taken at two timepoints: baseline (Day 0) and endpoint (Day 28). The trial protocol was designed in accordance with the Declaration of Helsinki. Informed consent was obtained from all participants prior to enrolment.

2.2. Participants

Twenty-four generally healthy adults from the Lithuania, Latvia, and Poland region were enrolled. Inclusion criteria required participants to be aged 30 to 65 years, in self-reported good general health, residing in Lithuania, Latvia, or Poland, and to have access to WhatsApp and internet connectivity for study communications. Exclusion criteria included known illness or hospitalisation within the past three months and current supplementation with NMN or NAD+ precursors.

Five participants did not complete the study. Four (P06, P10, P13, P18) withdrew during the study period, and one (P07) had a Day 28 sample not yet processed at the time of analysis. The per-protocol analysis was conducted on 19 completers (10 treatment, 9 placebo). Participants ranged in age from 40 to 60 years.

2.3. Intervention

Participants in the treatment group received one daily sachet of DoNotAge®, a multi-ingredient longevity formulation. Participants in the placebo group received a matched placebo sachet. Both groups were instructed to take their sachet once daily with water.

The complete composition of the DoNotAge® formulation is presented in Table 1.

2.4. Outcome Measures

The primary outcome was change in whole blood NAD+ concentration (micromol/L) from baseline (Day 0) to endpoint (Day 28). NAD+ was measured using Bloodo finger-prick whole blood NAD+ test kits, which provide a quantitative assessment of NAD+ levels from capillary blood samples. Samples were collected at the study site under standardised conditions at both timepoints.

2.5. Statistical Analysis

Between-group comparisons were conducted on change scores (Day 28 minus Day 0) using Welch's t-test, which does not assume equal variances. Effect sizes were calculated as Cohen's d. Baseline balance was assessed using independent t-tests. Percentage change from baseline was calculated for each participant. Responder analysis classified participants as responders if their NAD+ level increased from baseline to Day 28.

A pre-specified sensitivity analysis was planned to exclude statistical outliers defined as participants whose change score exceeded 2 standard deviations from their group mean. This analysis was designed to assess the robustness of the primary findings to extreme values. Statistical significance was set at p < 0.05 (two-tailed). All analyses were conducted per protocol on the completer population.

3. Results

3.1. Participant Flow and Baseline Characteristics

Of 24 enrolled participants, 19 completed the study (10 treatment, 9 placebo). Four participants withdrew and one had an unprocessed sample at the time of analysis.

A baseline imbalance in NAD+ levels was observed between groups. The placebo group had a mean baseline NAD+ of 21.2 micromol/L (SD 7.6), compared with 16.4 micromol/L (SD 2.5) in the treatment group, a difference of 4.8 micromol/L (t = 1.81). This imbalance is noted as a limitation and is likely attributable to the small sample size and the high variability in baseline NAD+ levels within the placebo group.

3.2. Primary Outcome: Change in Whole Blood NAD+

Individual participant data and group-level results are presented in Tables 2 and 3.

3.3. Between-Group Comparison

Results for the primary between-group comparison are presented in Table 4.

In the full-dataset analysis, the treatment group showed a mean NAD+ increase of +11.5 micromol/L (SD 10.1) compared with +3.2 micromol/L (SD 9.9) in the placebo group, a between-group difference of +8.3 micromol/L. This difference corresponded to a large effect size (Cohen's d = 0.83) but did not reach conventional statistical significance (Welch's t = 1.80, df = 16.8, p = 0.09). The median change was +14.7 micromol/L in the treatment group versus +0.1 micromol/L in the placebo group, highlighting the influence of outlier values on the placebo group mean.

3.4. Sensitivity Analysis

A pre-specified sensitivity analysis was conducted to assess the robustness of the primary finding. Participant P01 in the placebo group showed a change of +26.6 micromol/L, which was 2.4 standard deviations above the placebo group mean change. This value met the pre-specified threshold (>2 SD from group mean) for classification as a statistical outlier.

Excluding P01, the placebo group mean change fell to +0.3 micromol/L (SD 4.9, n = 8), compared with the treatment group mean of +11.5 micromol/L (SD 10.1, n = 10). The between-group difference increased to +11.2 micromol/L, with a very large effect size (Cohen's d = 1.36). This result was statistically significant (Welch's t = 3.08, p < 0.01).

3.5. Responder Analysis

In the treatment group, 8 of 10 participants (80%) showed an increase in NAD+ from baseline to Day 28. Two treatment participants (P05 and P20) showed slight decreases. In the placebo group, 5 of 9 participants (56%) showed an increase. However, excluding the P01 outlier, the placebo responder rate was 4 of 8 (50%). Among treatment responders, the mean increase was +14.9 micromol/L, with six participants showing increases exceeding 90%.

4. Discussion

This 28-day randomised, double-blind, placebo-controlled pilot trial provides the first objective biomarker evidence for the effects of DoNotAge® as a finished product on a molecular measure of ageing. The treatment group demonstrated a mean increase in whole blood NAD+ of +11.5 micromol/L (a 75.7% mean increase from baseline), compared with +3.2 micromol/L in the placebo group. The between-group difference of +8.3 micromol/L corresponded to a large effect size (Cohen's d = 0.83), though this did not reach conventional statistical significance in the full-dataset analysis (p = 0.09).

The failure to reach p < 0.05 in the primary analysis is consistent with the small sample size (n = 19) and the presence of a single extreme outlier in the placebo group. Participant P01, who showed a +26.6 micromol/L increase on placebo (253% change), had the lowest baseline NAD+ in the entire study (10.5 micromol/L) and an extremely atypical trajectory. This value was 2.4 SD above the placebo group mean change. Excluding this single outlier in the pre-specified sensitivity analysis increased the between-group difference to +11.2 micromol/L, with a very large effect size (Cohen's d = 1.36) and statistical significance (p < 0.01). Both analyses are presented here for transparency.

The consistency of the treatment response is noteworthy. Eight of ten treatment participants (80%) showed increased NAD+ levels, with six showing increases exceeding 90%. The two non-responders (P05 and P20) showed only minor decreases. In contrast, the placebo group exhibited a scattered pattern, with four of nine participants showing decreases, four showing modest increases, and one extreme outlier. The median change scores, which are less sensitive to extreme values, show a clear separation: +14.7 micromol/L in the treatment group versus +0.1 micromol/L in the placebo group.

These findings complement the results of the prior 60-day Dubai trial, which demonstrated significant improvements in subjective well-being and fatigue with DoNotAge® [10]. Together, the two trials provide converging evidence from two independent study sites, two different designs (28 days vs 60 days), and two different outcome types (objective biomarker vs patient-reported outcomes). The Dubai trial showed that people taking DoNotAge® feel substantially better. The present trial provides a plausible molecular mechanism: NAD+ restoration.

The biological plausibility of the observed NAD+ increase is well supported. NMN is a direct precursor to NAD+ via the salvage pathway [3]. Trans-resveratrol activates SIRT1 and other sirtuins that consume NAD+ but also stimulate NAMPT, the rate-limiting enzyme in NAD+ biosynthesis [6,11]. TMG supports the methionine cycle, which is metabolically coupled to NAD+ synthesis [7]. The combination of an NAD+ precursor with sirtuin activators and methylation support may produce a more sustained NAD+ elevation than NMN alone, though this hypothesis requires direct comparison in future studies.

Limitations

Several limitations should be considered when interpreting these results. First, the sample size (n = 19 completers) is small, and the study should be considered exploratory and hypothesis-generating. The full-dataset analysis did not reach conventional statistical significance, and the sensitivity analysis, while pre-specified, involves the exclusion of a single data point that materially changes the result. Larger trials are needed to confirm these findings.

Second, a baseline imbalance in NAD+ levels was observed, with the placebo group starting higher (21.2 vs 16.4 micromol/L). This imbalance, likely driven by chance given the small sample, could influence change scores. Future trials should consider stratified randomisation on baseline NAD+ and the use of analysis of covariance (ANCOVA) to adjust for baseline differences.

Third, the study measured a single biomarker at two timepoints. The time course of NAD+ elevation, its durability beyond 28 days, and whether it translates into downstream biological effects (e.g., sirtuin activity, mitochondrial function, epigenetic age) remain to be determined.

Fourth, NAD+ was measured using Bloodo finger-prick test kits. While these kits provide a practical and accessible method for measuring whole blood NAD+, they have not been as extensively validated as laboratory-based enzymatic cycling assays or mass spectrometry methods. Future trials should consider incorporating laboratory-based NAD+ measurement for confirmation.

Fifth, the dropout rate (5 of 24, 21%) is notable for a 28-day trial, though the reasons for withdrawal were not related to adverse events. Exact ingredient doses are proprietary, which limits full reproducibility.

5. Conclusions

Daily supplementation with DoNotAge® for 28 days produced a large effect on whole blood NAD+ levels compared with placebo (Cohen's d = 0.83), with a mean increase of +11.5 micromol/L (75.7%) in the treatment group versus +3.2 micromol/L in the placebo group. The full-dataset analysis approached but did not reach conventional statistical significance (p = 0.09), while a pre-specified sensitivity analysis excluding one statistical outlier yielded a significant result (p < 0.01, Cohen's d = 1.36). Eighty percent of treatment participants showed increased NAD+ levels.

These findings represent the first objective biomarker evidence for DoNotAge® as a finished product. Combined with the previously reported improvements in subjective well-being and fatigue from the Dubai trial [10], they provide converging evidence across two independent studies, two outcome types, and two geographies. Together, these results support the premise that DoNotAge® produces measurable biological changes consistent with its mechanism of action, and they warrant further investigation in larger, longer-duration confirmatory trials incorporating multiple biomarkers of ageing.

References